The Adeno-associated virus recombinant AAV (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, two major challenges that must be overcome for full realization are transient or low transgene expression and the detrimental effect of circulating host antibodies. The primary goal of our research is to use structural biology approaches, combined with biochemistry, molecular biology, and in vivo animal studies to develop AAV viral vectors that overcome these challenges and have improved clinical therapeutic efficacy. A key component to success is to identify regions of the capsid that participate in cellular interactions leading to or altering infection, i particular cell receptor attachment and interaction with host immunity, especially neutralizing antibodies. In this project, we will dissect the determinants of tissue transduction and also determine if these overlap with antigenic regions of the capsid. To get a general understanding we will compare AAV1/6, AAV5, AAV8, and AAV9, which represent the range of sequence and structure diversity among the AAV serotypes and have shown promise for clinical applications. We will take a reductionist approach to tackle the problem. We will first use structural biology, namely X-ray crystallography and cryo-electron microscopy and image reconstruction, to define the most antigenically reactive regions of the AAV capsids and then determine their role in infection through molecular and biochemical methods. The goal will then be to identify and mutate the residues that do not affect infection, characterize resulting vectors with respect to their antigenic reactivity against human serum, including those from post-AAV clinical gene therapy patients, and use the data arising as the guiding principle for general antibody escape AAV vector design. These will be tested in vivo, including the use of a clinically relevant hemophilia B mouse model. The overall impact of this project will be an understanding of the AAV capsid determinants necessary for successful infection and the neutralization of this process by host antibodies, a fundamental goal of virology. This information is critical for the gene therapy community in order to engineer recombinant AAV vectors for successful gene delivery in the presence of a host immune response, a natural host defense mechanism that cannot be prevented but must be circumvented in gene delivery applications because it dramatically reduces or eliminates gene expression.